Polymorphism in intron 4 of HFE does not compromise haemochromatosis mutation results

Two recent reports have described a polymorphism in HFE in the binding region of a PCR Primer Widely used in diagnosis of hereditary haemochromatosis (HHC), This polymorphism, IVS4+48G/A, is associated with the wild-type allele at the site of the common haemochromatosis mutation C282Y (845G arrow right A), and may prevent amplification of this allele. In population screening of 5,211 voluntary blood donors, Jeffrey et al. found that this polymorphism caused 15 C282Y/IVS4+48G/A compound heterozygotes to be incorrectly assigned as C282Y homozygotes. In a regional referral laboratory, Somerville et al. reported that 8 of 202 referrals were incorrectly assigned as C282Y homozygotes. We have retyped 944 samples for the C282Y mutation by a new primer external to IVS4+48G/A or by sequencing. We confirmed 575 previously diagnosed C282Y homozygotes using the new primer, as well as 192 C282Y wild-type homozygotes, including 10 IVS4+48G/A homozygotes, and 177 heterozygotes. Of the heterozygotes, 28 were C282Y/IVS4+48G/A compound heterozygotes which had been correctly reported using the original Feder reverse primer. Three of these, however, had a mutant:wild-type band ratio of approximately 5:1. Re-extraction of DNA from these samples resulted in bands of expected intensity for heterozygotes. This indicates that DNA purity may have an effect on accuracy of diagnosis. We did not observe non-amplification of the polymorphic allele under our various standard PCR conditions (http://genetics.nature.com/supplementary_info/).